Abstract: Borrelia burgdorferi (Bb), the Lyme disease spirochete, undergoes dramatic adaptive changes as it cycles in nature between its diverse tick and mammalian hosts. Prior to work conducted by the PI over the past funding interval, little was known regarding the genetic regulatory networks that modulate Bb's adaptive changes contributing to Bb's infectivity and virulence. To this end, we discovered a novel regulatory pathway in Bb, wherein one alternative sigma factor ([unreadable]N, RpoN) regulates the expression of another alternative sigma factor ([unreadable]s, RpoS) which, in turn, governs the expression of key membrane lipoproteins associated with Bb's virulence. The pathway is controlled by an enhancer-binding protein, response regulator protein-2 (Rrp2), which first must be activated for the subsequent activation of RpoN and the consequent transcription of rpoS. Although the discovery of this regulatory network in Bb has represented an important advance in the Lyme disease field, it also has prompted many new questions regarding its direct and indirect regulatory effects, how the pathway intersects with other regulatory mechanisms, and how and to what extents various genes of Bb are influenced. Selected areas of investigation over the next funding interval will continue to address some of these salient information gaps. Accordingly, the Specific Aims of this proposal are: (1) To discern those genes directly influenced by the RpoN- RpoS pathway. We shall combine our expertise in Bb microarrays with our newly developed lac repressor/operator system to artificially control the expression of rpoS in Bb. Identified genes will become the subject of further studies (Aim 4);(2) To investigate the mechanism by which the RpoN-RpoS pathway regulates atypical expression of the decorin-binding protein (DbpB/A) operon. The dbpBA operon is under the control of the RpoN-RpoS pathway, but it has an expression pattern different from other genes controlled by RpoS (e.g., ospC). As such, dbpBA may be a model of how certain genes influenced by the pathway also are subjected to another layer of regulation. Emphasis will be placed on analyzing key features of the dbpBA promoter and upstream cis-elements;(3) To examine the expression of rpoS over the complete enzootic life cycle (tick and mammalian phases) of Bb;and (4) To examine the phenotypes of Bb mutants deficient in selected RpoS-dependent genes. These efforts will provide an expanding foundation for further characterizing the novel RpoN-RpoS regulatory network in Bb, and for potentially identifying regulatory and virulence-associated genes that contribute to many aspects of Bb's complex parasitic strategy. Understanding the roles of these genes could lead to new intervention strategies (vaccines, therapeutics) for Lyme disease.